Titin and nebulin are unprecedented giant proteins discovered and named in this laboratory. Our recent work has focused on the roles of titin and nebulin in the structure, regulation, mechanics and assembly of sarcomeres in striated muscles. We have employed a multidisciplinary approach that draws on methodologies and concepts of biochemistry, biophysics, cell and molecular biology, and physiology. (A). Titin: A primary theme of our research is the understanding of the structural and physiological roles of titin, a family of giant structural proteins that constitute an elastic matrix in the striated muscle sarcomeres and some nonmuscle cells. Titin and the elastic matrix may play major physiological roles, including the genesis of long range elasticity, the maintenance of sarcomere stability, the assembly of nascent sarcomeres in developing muscle cells, and the assembly of myosin in the microfilaments in nonmuscle cells. The following questions of central importance are being addressed: 1.How do titin filaments respond to stretch and release? We are refining our working hypothesis that the segmental extensibility of titin underlies its capacity as a multistage molecular spring. Site-specific monoclonal antibodies to various titin sequence domains have been prepared and applied in immunoelectron microscopy to delineate the order, locus and degree of titin extension in skeletal, cardiac and flight muscles. 2. What is the molecular basis of titin elasticity? We are testing the hypothesis that specific and reversible unfolding of domains and uncoiling of titin filaments underlies its capacity as a molecular spring. The elasticity of isolated titin and analogous elastic molecules from skeletal and cardiac and flight muscles are being measured directly, as single molecules and as gel networks, with laser trap techniques, atomic force microscopy and rheometric techniques. The conformation of titin domains and synthetic peptides will be studied by high resolution NMR and X-ray crystallography. Titin elasticity will be explained in the context of statistical theories of polymers and realistic structural features of titin domains. 3.Do thin filaments modulate titin elasticity? We are searching for transient or stable interactions of titin with actin, tropomyosin, troponin, nebulin and other thin filament proteins. Mechanical measurements will be done on muscles in which actins in thin filaments have been selectively removed by gelsolin as well as on fibers that have been reconstituted with selected regulatory proteins to evaluate the thin filament's contribution to elasticity in situ. 4. Does titin participate in the regulation of actin-myosin interactions? The effect of titin on the actin-myosin interactions in solution will be evaluated by ATPase activity, in vitro motility assays, and molecular force measurements. In the past year, we have succeeded in revealing, for the first time, that the elastic PEVK segment of titin is modular in construction, display repeating proline II helix /coil conformational motifs and most importantly, interact with thin filaments in a calcium/S100 sensitive manner. These observations established the conformational basis of titin PEVK elasticity and identified PEVK as a site of interfilament interaction and that S100 or calcium sensor proteins regulate interaction and titin elasticity. In this past year, we have discovered that titin PREV segment is a giant force sensing molecule of the SH3 signaling pathways. High resolution structures of a homopolymer of a PEVK module in solution and in gel phase has been determined by NMR techniques. The extensive differential splicing of titin PEVK exons in various human heart muscles was elucidated. (B). Nebulin: Nebulin is thought to act as a protein ruler that regulates the length of actin filaments in skeletal muscle sarcomeres. Our recent studies indicate that nebulin tethers myosin heads to actin in a calcium/calmodulin sensitive manner, raising the intriguing possibility that nebulin may act as a calcium-linked regulatory protein that is distinct from the classic troponin/tropomyosin system. 1. How does nebulin regulate the length of thin filaments? The length, mass and sequence of nebulin size isoforms will be compared with thin filament lengths of a wide range of skeletal muscles in developing and adult muscles. In particular, we will test the hypothesis that both the number of nebulin repeats and the presence of a specific N-terminal termination sequence are necessary to give rise to uniform thin filament length distribution within each sarcomere. The effect of purified native nebulin on the length distribution of polymerizing or performed actin filaments will be monitored by electron microscopy and fluorescence microscopy to test the length regulating ability of nebulin and other potential cofactors. At a later stage, chimeric constructs consisting of sequence motifs from distinct nebulin will be produced and assayed to fine-tune their length regulating property. 2. How does nebulin regulate actin-myosin interaction? The kinetics of nebulin on ATPase activities will be studied by stop flow methods to define plausible molecular mechanisms of action. The influence of nebulin and calcium/calmodulin on the sliding velocity of actin over myosin will be investigated by in vitro motility assays to reveal the effect of nebulin on mechanical coupling of myosin motors. The search for other potential calcium mediators will be done by affinity chromatography and blotting. We are particularly interested in the possible involvement of S100 proteins as well as the kinase/phosphotase that phosphorylate nebulin as physiological effectors of actin/myosin interaction. Single muscle mechanics will be carried out to test if nebulin affects the shortening velocity or resting stiffness of skeletal muscles. Ultimately, calcium regulation of active tension, active stiffness and shortening velocity will be studied in muscle fibers that are reconstituted with distinct nebulin isoforms or chimeras. We have succeeded in purifying full-length native nebulin as well as nebulin-containing native thin filaments. Collaborative efforts have been initiated to apply cryoEM techniques to study the 3D images of such "composite" thin filaments, with and without myosin decoration. These studies are beginning to reveal if and how nebulin participates in the steric blocking of myosin on actin. Additionally, atomic force microscopy has been used to study both the morphology as well as the molecular forces of interactions between actin, myosin and nebulin. The synthesis and incorporation of titin, nebulin/nebulette into sarcomeres of developing skeletal and cardiac muscle cells in culture has being examined with immunofluorescence confocal microscopy. Nonmuscle nebulin family members such as cortactin and LASP are being investigated for their roles in cell motility.